Introduction

Cyclic nucleotide PDEs hydrolyze and inactivate cAMP. PDE genes are divided into 11 families. Because individual families can contain as many as 4 genes that can be processed to yield multiple transcripts, most cells express numerous PDEs. Recent studies have established that certain PDEs can selectively interact with other cellular proteins, and assemble into specialized macromolecular complexes within discrete functional compartments, thereby controlling local cellular cAMP-mediated, PKA-dependent signaling.1,2 The heart can express PDEs from each of the PDE1 through PDE5 families, as well as the PDE8 family.3 Because reductions in cAMP/PKA signaling contribute to impaired cardiac function in heart disease patients,4 PDE inhibitors were initially promoted for treating heart failure. However, despite their benefit in treating contractile failure, prolonged treatment of heart failure patients with PDE inhibitors increases mortality, principally by promoting sudden cardiac death.5 Selective targeting of PDE isoenzymes may provide novel opportunities to correct the impaired cAMP-dependent signaling seen in heart disease without these adverse consequences.

Consistent with current paradigms of cAMP compartmentalization, PDE3 and PDE4 enzymes suppress basal cAMP/PKA signaling and contractility in cellular microdomains containing SR Ca2+pumps but not RyR2s or L-type Ca2+channels.6,7 Because PDE4D isoforms associate with SERCA2a7 and RyR2s,8 we investigated PDE4D's role in regulating cardiac contractility. Mice lacking PDE4D have enhanced baseline cardiac contractility associated with increased PLN phosphorylation, SR Ca2+ content, and Ca2+ transients but not elevated ICa,L or RyR2 phosphorylation. PDE4D also coassembles with SERCA2a but not with RyR2 in both murine and human hearts.

Methods

PDE4D-deficient (PDE4D−/−)9 and littermate WT mice (129vj/c57 background) were studied at 10 to 12 weeks of age. Experiments were conducted in accordance with the Canadian Council of Animal Care. Detailed methods are in the Online Data Supplement at http://circres.ahajournals.org.

Ca2+ transients and ICa,L measurements. A, Ca2+ transients (upper) and ICa,L (lower) recorded for WT and PDE4D−/− cardiomyocytes in response to voltage steps (indicated) from −85 mV holding potential and a 500-ms prepulse ramp to −45 mV. B, Typical Ca2+ transients and ICa,L at +10 mV before and after ROL application. C and D, Mean Ca2+ transient and ICa,L peaks as a function of voltage in the presence and absence of Rp-cAMP (C) and ROL (D). *P<0.01 vs control within same group; †P<0.01 vs WT control.

Compared with WT, PDE4D−/− myocardium had elevated (P<0.05) PLN phosphorylation levels (Figure 3), which were indistinguishable from ROL-treated WT myocardium, as well as either ROL-treated or ROL-untreated PDE4D−/− myocardium (not shown). In contrast, RyR2 phosphorylation (Figure 3) was not different (P=0.11) at serine 2030, the PKA-dependent site regulating function,11 or S2814 (P=0.79), but was unexpectedly8 decreased (8%, P=0.01) at S2808. Coimmunoprecipitation in preparations from mouse and human myocardium revealed that PDE4D associates with SERCA2a but not RyR2 (Figure 4). Although PI3Kγ is required for PDE4 activity in microdomains containing SERCA2a,8 it was not detected in SERCA2a immunoprecipitates, suggesting that PI3Kγ's enzymatic activity6,7,12 is required. This is consistent with elevated contractility seen in Langendorff hearts treated with the PI3K inhibitor wortmannin (Online Figure VI).

PDE4D interactions with SERCA2a in murine and human myocardium. A, Representative Western blots (repeated in 3 separate hearts) probing with PDE4D in heart lysates from a PDE4D−/− and WT mouse heart homogenates that were immunoprecipitated (IP) using control IgG or using RyR2- or SERCA2a-specific antibodies. Results show that PDE4D antibodies recognized bands at molecular weights of 97 kDa (corresponding to PDE4D-3, -7, and -9 splice variants) in immunoprecipitation reactions with anti-SERCA2a antibodies, but not with anti-RYR2 antibodies or with IgG controls. A very weak nonspecific band having a molecular weight of ≈110 kDa was detected in all immunoprecipitation groups, as well as in homogenates from the PDE4D-null mice. B, Representative inputs for immunoprecipitation reactions shown in A. C, Results of SERCA2a immunoprecipitation in human hearts. D, Diagram summarizing implications of our results in WT (left) and PDE4D−/− (right) myocardium.

Selective inhibition of PDE414 elevated contractility, Ca2+ transients, Ca2+ SR loads, and PLN phosphorylation in WT hearts, as reported,7 to levels seen in PDE4D−/− myocardium without affecting PDE4D−/−. Thus, although PDE4A and PDE4B are expressed in mouse heart,15 PDE4D underlies the cAMP-dependent baseline contractile responses to PDE4 inhibitors. Although PDE4D ablation does not induce expression changes of other PDE isoforms in different organs,9 changes in activity/expression of other PDE isozymes might have occurred. In this regard, responses to PDE3 inhibitors (which also affect mouse baseline contractility7,16) were unaffected by PDE4D ablation (Online Figure V). Although inhibition of PDEs from other families was not examined, the consequences of PDE4D ablation on cardiac function can be readily explained by the loss of PDE4D activity alone.

A previous study reported that PDE4D and RyR2 interact in murine cardiomyocytes and that PDE4D−/− mice develop both heart dysfunction and arrhythmias by 9 months of age resulting from RyR2 hyperphosphorylation at Ser2808, which we did not find. These differences may be related to differences in reagents used in the 2 studies or to age-dependent changes in cAMP regulation or heart function.17 In this regard, we did observe declines in cardiac function of PDE4D−/− mice at 9 months,8 which helps explain why another study,15 focusing on β-adrenergic responses, only identified acceleration of Ca2+ transient relaxation in PDE4D−/− myocytes at 5 to 6 months, accompanied by trends (nonsignificant) toward elevations of Ca2+ transients, as well as myocyte shortening/relaxation (discussed further in the Online Supplement).

Age-dependent deterioration of PDE4D−/− heart function is not inconsistent with human data showing that persistent cAMP-dependent stimulation with, for example, PDE3 inhibitors promotes disease progression, mortality, and arrhythmias in heart disease patients, despite providing short-term benefit (by enhancing contractility) in transplant and heart failure patients.18 On the other hand, clinical trials with PDE4 inhibitors did not identify cardiovascular side effects (besides slight increases in atrial fibrillation).19 These differences between mouse and humans may arise from higher relative PDE4 activities in mouse (≈35%) versus human (<10%) myocardium.20 However, PDE activity is highly compartmentalized,1 making local (not global) activity most relevant functionally. Thus, PDE4D tethering to SERCA2a combined with the high PDE4 activity (≈50% of total activity) observed in PLN immunoprecipitates from human myocardium20 suggests that PDE4D fine-tunes the cAMP-dependent SR Ca2+-ATPase activity in human hearts.20 This observation is particularly relevant because heart disease or failure is invariably associated with impairment of cAMP/PKA-dependent SR Ca2+-ATPase activity. Moreover, SERCA2a gene therapy21 or β-blocker treatment (which enhances SERCA2a expression22) improves heart function and longevity. Thus, our findings support the possibility that selective PDE4D inhibition could improve contractility in diseased hearts by specifically elevating cAMP in SR microdomains containing SERCA2a/PLN. Remarkably, there is a paucity of definitive data available on direct actions of PDE4 inhibitors on cardiac function in humans or in animal models. Clearly, more studies are required to fully determine the role of PDE4D and its inhibition in heart disease patients.

Sources of Funding

This work was supported by a Canadian Institutes of Health Research grant to Dr Backx, who is a Career Investigator with the Heart and Stroke Foundation (HSF) of Ontario. Dr Beca holds a postdoctoral fellowship from the Heart and Stroke Richard Lewar Centre of Excellence (HSRLCE), University of Toronto. Dr Helli held postdoctoral fellowships from HSRLCE and HSF of Canada. Support was also provided by Foundation Leducq Grant 06CVD02 cycAMP (to M.C. and M.A.M.), National Institutes of Health Grant HL0927088 (to M.C.), and grants from the United States Department of Veterans Affairs Medical Research Funds (to M.A.M.).

Disclosures

None.

Footnotes

In July 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.5 days.

. Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon β-adrenergic stimulation in normal and failing hearts. Biochem J. 2006;396:7–16.

. The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors. Biochem J. 1987;241:535–541.

What New Information Does This Article Contribute?

Although PDE inhibitors can improve cardiac function, their prolonged use is associated with heart-related morbidity and mortality. Given the diversity of PDE isozymes, precise targeting of selected PDE isoforms is predicted to enable pinpoint modulation cAMP-dependent signaling in cellular microdomains. Indeed, we show that genetic elimination of PDE4D (1 of 4 genes in the PDE4 family) elevates cAMP levels in subcellular compartments containing the SR Ca2+ pump (ie, SERCA2a/phospholamban), but not L-type Ca2+ channels or cardiac ryanodine receptors, leading to elevations in phospholamban phosphorylation, SR Ca2+ levels, whole-cell Ca2+ transients, and cardiac contractility. These data show that, in principle, selective pharmacological targeting of PDE4D in SR microdomains allows precise pharmacological control of cardiac contractility, while potentially minimizing the side effects associated with broader spectrum inhibition. These findings might have significant implications in developing treatment strategies for heart disease patients.